SMM ID4 Measured in Biochemical System Using Small Molecule MicroArray - 2128-01_Other_SinglePoint_HTS_Activity

Small Molecule Microarrays (SMM) are generated by covalently immobilizing small molecules on an isocyanate surface glass slide. SMMs are incubated with the Flag-tagged ID4 (Id-related helix-loop-helix protein ) target protein solution at 4 degrees for 2 hours. The slides are then breifly washed and incubated with an anti-His Flag antibody. Slides are breifly washed, and fluroescent anti-rabbit more ..

Keywords: ID4, F/B, SMMAssay Overview:Small Molecule Microarrays (SMM) are generated by covalently immobilizing small molecules on an isocyanate surface glass slide. SMMs are incubated with the Flag-tagged ID4 (Id-related helix-loop-helix protein ) target protein solution at 4 degrees for 2 hours. The slides are then breifly washed and incubated with an anti-His Flag antibody. Slides are breifly washed, and fluroescent anti-rabbit antibody was incubated with slides. Slides arewashed dried ans scanned. Fluorescence on each spot is monitored and a spot that exhibits a Fluorecence over Background (F/B) with a P-value less than 7% is considered a positive. Each compound is printed 4 times on the slide and compounds that return F/B with P-values <7% 4 times are considered active and are therefore labelled as hits.

Expected outcome:increase in fluorescence upon portein binding to compounds on slides

Slide Manufacturing:Gamma-aminopropylsilane (GAPS) slides (Corning) were incubated in a solution of FMOC-8 amino-3,6,dioctanoic acid (10mM), PyBOP (10mM), and diisopropylethyl amine (20mM) in DMF for 4 hr. The slides were washed in DMF and incubated in 2% (v/v) piperidine in DMF for 10 min to remove the FMOC group from the surface. Following a rinse with DMF, the slides were activated in a solution of 1% (v/v) 1,6-diisocyanatohexane in DMF for 30 min. Slides were thoroughly rinsed with DMF then THF and dried with a stream of N2 gas. 200pL of compounds were then printed on the slides with a robotic microarrayer (Aushon 2470) using 5ul 10mM solutions of compounds in DMSO. After arraying, slides were placed in a desiccator for at least 16 hrs. with an open vial of pyridine, and a mild vacuum was pulled to create a pyridine vapor atmosphere. The slides were then immersed in a solution of (PEO)3-monoamine (10mM) in DMF to quench any un-reacted isocyanate functional groups on the slide surface. The slides were then thoroughly rinsed with DMF and THF then dried with a stream of N2 gas and stored under argon gas until needed for binding assays.Assay Protocol:1. STAT-3-was diluted to 1ug/ml in Tris buffer saline with tween (TBST) containing: 50mM Tris, 150mM NaCl, 0.1% Tween,3mM DTT and 0.5mM EDTA.2. 3ml of STAT3 solution was added to each of the slide and allowed to Rock at 4degrees for 2hours3. Slides were rinsed 2X with TBST.4. 1/5000 dilution Rabbit Anti-flag Antibody was prepared in TBST to a final concentration of 0.1ug/ml.5. 3ml of primary antibody was added to the slides for 30 minutes at RT.6. 1/5000 dilution of conjucated anti-Rabbit-IgG was prepared in TBST toa final concentation of 0.1ug/ml 7. Slides were rinsed 2X in TBST and 1X with H2O8. Slides are dried and scanned using Genepix at 500 gain.

PRESENCE OF CONTROLS: Neutral control wells (NC) were included on every plate. EXPECTED OUTCOME: Active compounds result in increasing readout signal. NORMALIZATION: No normalization was applied to the raw data signals. PATTERN CORRECTION: No plate pattern correction algorithm from Genedata Condoseo (v.7.0.3) was applied. PUBCHEM_ACTIVITY_SCORE: Each data point was assigned a P-value based on a Cauchy distribution of a block of spots on an array (220 spots per block). The activity threshold for the P-value was set at 7%. Compounds were considered active if all replicates (n=4) passed the activity threshold. Compounds called active based on this criterion were assigned a Pubchem_Activity_Score of 100; compounds that were not active were assigned a Pubchem_Activity_Score of 0. PUBCHEM_ACTIVITY_OUTCOME: Active sampels were assigned an outcome of 2 (active): PUBCHEM_ACTIVITY_SCORE = 100 Inactive samples were assigned an outcome of 1 (inactive): PUBCHEM_ACTIVITY_SCORE = 0

Each data point was assigned a P-value based on a Cauchy distribution of a block of spots on an array (220 spots per block). The activity threshold for the P-value was set at 7%. Compounds were considered active if all replicates (n=4) passed the activity threshold. Compounds called active based on this criterion were assigned an Activity_Score of 100; compounds that were not active were assigned an Activity_Score of 0.